Renogrit selectively protects against cisplatin-induced injury in human renal tubular cells and in Caenorhabditis elegans by harmonizing apoptosis and mitophagy

Cisplatin-induced nephrotoxicity restricts its clinical use against solid tumors. The present study elucidated the pharmacological effects of Renogrit, a plant-derived prescription medicine, using cisplatin-induced human renal proximal tubular (HK-2) cells and Caenorhabditis elegans. Quantification of phytochemicals in Renogrit was performed on HPTLC and UHPLC platforms. Renogrit was assessed in vitro in HK-2 cells post-exposure to clinically relevant concentration of cisplatin. It was observed that renoprotective properties of Renogrit against cisplatin-induced injury stem from its ability to regulate renal injury markers (KIM-1, NAG levels; NGAL mRNA expression), redox imbalance (ROS generation; GST levels), and mitochondrial dysfunction (mitochondrial membrane potential; SKN-1, HSP-60 expression). Renogrit was also found to modulate apoptosis (EGL-1 mRNA expression; protein levels of p-ERK, p-JNK, p-p38, c-PARP1), necroptosis (intracellular calcium accumulation; RIPK1, RIPK3, MLKL mRNA expression), mitophagy (lysosome population; mRNA expression of PINK1, PDR1; protein levels of p-PINK1, LC3B), and inflammation (IL-1β activity; protein levels of LXR-α). More importantly, Renogrit treatment did not hamper normal anti-proliferative effects of cisplatin as observed from cytotoxicity analysis on MCF-7, A549, SiHa, and T24 human cancer cells. Taken together, Renogrit could be a potential clinical candidate to mitigate cisplatin-induced nephrotoxicity without compromising the anti-neoplastic properties of cisplatin.


Renogrit treatment decreased cell injury in cisplatin-induced HK-2 cells
Treatment of HK-2 cells with Renogrit (3-300 µg/ml) for 24 h showed no significant change in cell viability (Fig. 3a) compared to untreated control (UC).Cisplatin (5-25 µg/ml) induced HK-2 cells showed a significant (p < 0.001) concentration-dependent decline in cell viability compared to UC (Fig. 3b).Cisplatin at 15 µg/ml was used for further analysis as it is a clinically relevant dose of cisplatin (C max of cisplatin as detected from the blood plasma of patients) 24 .Renogrit (3-100 µg/ml) treatment led to a concentration-dependent rise in the viability of cisplatin-induced HK-2 cells which was significant in cells treated with 30 µg/ml (p < 0.01) and 100 µg/ml (p < 0.001) of Renogrit.NAC (2 mM) treated group showed a marginal improvement in cell viability (Fig. 3c).Later, the biomarkers of cisplatin-induced kidney injury namely NAG, KIM-1, and NGAL were evaluated 25 .It was observed that cisplatin-induced HK-2 cells showed a significant (p < 0.01) increase in NAG levels.Cisplatininduced cells in the presence of Renogrit (30 and 100 µg/ml) showed normalized levels of NAG release (Fig. 3d).The released KIM-1 levels were also significantly (p < 0.001) increased in cells induced with cisplatin compared to control.Cisplatin-induced HK-2 cells in presence of Renogrit (30 and 100 µg/ml) treatment showed a significant (p < 0.001) decline in released KIM-1 levels (Fig. 3e).Furthermore, Renogrit (10, 30, and 100 µg/ml) treated cells significantly (p < 0.001) decreased the overexpression of NGAL gene (Fig. 3f).NAC treatment of cisplatin-induced cells also led to the decline of NAG, KIM-1 release and NGAL gene upregulation (Fig. 3d-f).

Renogrit treatment reduced markers of oxidative stress in cisplatin-induced HK-2 cells
Real-time analysis of ROS generation up to 120 min showed that HK-2 cells treated with Renogrit (10, 30, and 100 µg/ml) displayed no production of ROS in response to cisplatin induction.However, cisplatin-induced cells showed a steady rise in the generation of ROS (Fig. 4a).The anti-oxidant activity of GST in kidney tissues is known to be suppressed by cisplatin 26 .Here, the significant (p < 0.01) decrease in the activity of GST enzyme in cisplatin-induced HK-2 cells got normalized in a concentration-dependent manner in the presence of Renogrit (Fig. 4b).An increase in ROS generation alters the mitochondrial membrane potential 27 .It was observed that cisplatin-induced cells showed a significant (p < 0.001) decrease in mitochondrial membrane potential as evaluated by ratiometric analysis of JC-1 dye aggregates.Renogrit-treated cells showed a significant (p < 0.01) increase in mitochondrial membrane potential compared to only cisplatin-induced cells (Fig. 4c).NAC (2 mM) treated cisplatin-stimulated cells also decreased the markers of oxidative stress (Fig. 4a-c).presence of Renogrit's marker compounds, GA (Rf = 0.36), MG (Rf = 0.52), QT (Rf = 0.57), and BG (Rf = 0.18) acquired at 280 nm using toluene: ethyl acetate: methanol: formic acid (5:4:1:0.2,by volume) as a developing system using densitometric scan.Quantitative analysis of the marker compounds in Renogrit was done based on the linearity plot and peak tables of (c) GA, (d) BG, (e) MG, and (f) QT along with RENO attained at 280 nm.Quantification of the phytochemicals has been mentioned in Table 1.

Renogrit treatment enhanced lysosome population and mitophagy in cisplatin-induced HK-2 cells
In renal tubular cells, cisplatin-induction is known to decrease lysosome numbers which leads to defects in mitophagy and eventually renal cell apoptosis 9,31 .In the current study also, cisplatin-induced HK-2 cells revealed  1. a significant (p < 0.01) decline in the lysosome population as observed from flow cytometry based analysis.When these cells were treated with Renogrit a concentration-dependent recovery of lysosome number was observed which was significant (p < 0.05) at 30 and 100 µg/ml concentration.NAC treatment also showed a noticeable rise in the lysosome population (Fig. 7a,b).The conversion of LC3B from LC3A and phosphorylation of PINK1 is essential for mitophagy 32 which was decreased in cisplatin-treated HK-2 cells.But in presence of Renogrit (100 µg/ml) their levels increased significantly (p < 0.05) (Fig. 7c,d).This also led to a reduction in the activation of cell death pathway as observed by the significant (p < 0.001) decrease in c-PARP1 levels in Renogrit-treated cells (Fig. 7e).Similar findings were obtained in NAC-treated cells (Fig. 7c-e).

Renogrit treatment reduced inflammation in human kidney cells
Pro-inflammatory cytokines, particularly IL-1β is associated with cisplatin-induced kidney injury and inflammation 33 .The activation of LXR-α is known to reduce cisplatin-induced renal inflammation 34 .Hence, in this study the effect of Renogrit on IL-1β and LXR-α was evaluated.HEK-Blue IL-1β reporter cells were used to evaluate the effect of Renogrit against stimulation of IL-1β signaling cascade.To rule out any confounding bias the cytosafety of Renogrit on HEK-Blue IL-1β reporter cells was evaluated.It was observed that Renogrit treatment did not affect the viability of HEK-Blue IL-1β reporter cells (Fig. 8a).Furthermore, in HEK-Blue IL-1β reporter cells stimulated with IL-1β (1 ng/ml), Renogrit (30 and 100 µg/ml) treatment significantly (p < 0.001) reduced the activity of IL-1β which was not observed in NAC (2 mM) treated cells (Fig. 8b).Also, Renogrit (100 µg/ml) treatment on cisplatin-induced HK-2 cells showed a significant (p < 0.05) increase in levels of LXR-α as compared to only cisplatin-treated cells (Fig. 8c).Collectively, Renogrit treatment might ameliorate cisplatin-induced renal injury and inflammation by modulation of IL-1β signalling cascade and LXR-α levels.

Renogrit treatment did not alter the anticancer property of cisplatin
In order to assess whether the presence of Renogrit hinders the anticancer potential of cisplatin four cancer cells derived from different organs were treated with cisplatin and Renogrit.Although, cisplatin induction significantly (p < 0.001) decreased the viability of MCF-7, A549, SiHa, and T24 cells, but Renogrit did not hinder the anticancer activity of cisplatin (Fig. 9a-d).Interestingly, Renogrit (100 µg/ml) treatment significantly (p < 0.01) decreased the viability of cisplatin-induced A549 cells (Fig. 9b).

elegans
Further validations of the pharmacological properties of Renogrit were performed on cisplatin-exposed C. elegans.Primarily, for the selection of optimum dose for treatment, nematodes were exposed to various concentrations of Renogrit and cisplatin.The progeny in cisplatin (20-100 µg/ml) treated N2 strain of nematode significantly (p < 0.001) decreased (Fig. 10a).Interestingly in Renogrit (300 µg/ml) treated worms a significant (p < 0.05) increase in population of progeny was observed (Fig. 10b).Based on these findings the optimum doses of Renogrit (10, 30, and 100 µg/ml) and cisplatin (40 µg/ml) were selected for further experiments.Upon Renogrit treatment on cisplatin exposed worms, the number of unhatched progenies significantly (p < 0.001) decreased (Fig. 10c).Cisplatin (40 µg/ml) exposure to N2 strain of nematodes led to a significant (p < 0.05) generation of ROS which decreased significantly (p < 0.001) in presence of Renogrit (10, 30, and 100 µg/ml) (Fig. 10d).Furthermore, it was observed that upon cisplatin (40 µg/ml) exposure to SYS81 strain of C. elegans, SKN-1 (mammalian homologue of NRF2) expression is enhanced which can be observed by the nuclear localization (green puncta formation) compared to untreated control (Fig. 10e).In presence of Renogrit (100 µg/ml)  the cisplatin-stimulated SYS81 worms have a similar expression of SKN-1 as untreated control.NAC (4 mM) treatment on the cisplatin-exposed worms also led to comparable findings.

Renogrit treatment enhanced lysosome population and mitophagy in cisplatin-exposed C. elegans
Similar to the observed modulations in the lysosome population in vitro, microscopy of cisplatin (40 µg/ml) treated N2 strain of worms showed that the lysosome number has declined as observed from the decrease in levels of LysoView 633 stain.But in the presence of Renogrit (100 µg/ml) the population of lysosomes was comparable to untreated control (Fig. 12a).PINK1 and PDR1 (nematode parkin homologue) are essential for mitophagy and maintenance of mitochondrial turnover 14,18,32 .Renogrit (100 µg/ml) treatment on cisplatin-exposed worms showed that the mRNA expression levels of PINK1 and PDR1 were significantly (p < 0.01) upregulated which declined upon cisplatin exposure (Fig. 12b,c).The mRNA expression levels of EGL1 increased significantly (p < 0.001) in cisplatin-exposed worms but with Renogrit (100 µg/ml) treatment the levels decreased in a significant (p < 0.05) manner.Similar, findings were observed in NAC (4 mM) treated groups.

Discussion
The current study aimed to analyze the phytopharmacological profile of the Ayurvedic prescription medicine Renogrit using in vitro and Caenorhabditis elegans based models.The phytometabolite characterization of Renogrit via HPTLC and UHPLC-based analysis showed the presence of Gallic acid, Bergenin, Methyl Gallate, Quercetin, and Boeravinone B as the major marker compounds.Natural variability in the phytochemical composition of botanical raw materials occurs in response to climate, harvest time, fertilization methods, storage conditions, and processing procedures.The difference in phytochemical composition can lead to variance in bioactivity of the herbal formulation.To ensure optimum bioactivity of Renogrit, three different batches were   analyzed for their phytochemical integrity.As observed, the phytochemicals in Renogrit of the three randomly analyzed batches had a similar phytochemical makeup.NAC was selected as a comparator for the entire study as several non-clinical and a clinical case have been reported wherein NAC was observed to act as a nephroprotective agent in cisplatin treated cell/animal/ patient [35][36][37][38][39] .The safety of NAC administration in patients with reduced renal function has also been established 40 .Furthermore, several studies have proposed that NAC has property to modulate oxidative stress, inflammation, apoptosis, and mitophagy [41][42][43][44] .Hence, NAC was used as an experimental control for this study.
Prior to the evaluation of bioactivity of Renogrit its effect on viability of HK-2 cells was evaluated.Renogrit was found to be cytosafe at all physiologically relevant concentrations.Moreover, for selection of the clinically relevant dose of cisplatin, its effect on viability of HK-2 was also evaluated.The concentration which showed cytotoxicity in 50% of the cell population and which was nearby its therapeutic concentration (Cisplatin at human C max ) 24 was utilized for further in vitro experiments.This allowed us to rule out any confounding bias in our obtained results.After observation of reversal in the cytotoxicity in cisplatin-induced HK-2 cells post Renogrit treatment biomarkers depicting renal tubular injury namely NAG, KIM-1, and NGAL 45 were also analyzed.It was observed that Renogrit-treated HK-2 cells were able to repel the cytotoxic effects of cisplatin, evident from the normalization of these molecular biomarkers of renal injury.This effect of Renogrit can be co-related with the presence of Gallic acid which was observed to act as a renoprotectant in cisplatin-induced rats 46 .
Oxidative stress and depolarization of mitochondrial membrane are one of the major molecular events linked with the pathophysiological manifestations of cisplatin-induced kidney injury 32 .Herein, when Renogrit was added along with cisplatin on HK-2 cells the generation of ROS was not observed.Further probing of the antioxidative properties of Renogrit showed that the activity of the antioxidant enzyme GST that decreased post cisplatin induction also got normalized.Herbal extracts and their isolated phytochemicals are known to enhance GST activity in animal models of cisplatin-induced kidney damage 47,48 .Mitochondria are abundantly present in renal tubular epithelial cells as their function of secretory reabsorption relies on mitochondrial oxidative phosphorylation.Oxidative stress in part promotes mitochondrial depolarization and dysfunction which leads to an imbalance in the normal functionality of renal tubular cells.In the presence of Renogrit, HK-2 cells were able to decrease the adverse effects of cisplatin-stimulated oxidative stress on mitochondrial membrane potential.The phytochemical Bergenin present in Renogrit is known to attenuate renal injury by resisting the change in mitochondrial dynamics, as observed in a rat model 49 .
Cisplatin stimulation is known to trigger several pathways of cell injury which leads to induction of apoptosis and cell death 50 .This is precisely the reason which makes cisplatin an indispensable anti-cancer agent but that also becomes a challenge to protect the healthy cells like normal renal tubular cells from innocent bystander death.Apoptosis is a type of programmed cell death which is mainly mediated by the caspase pathway wherein caspase-3 plays a primary role.Another major cellular event linked with promotion of cisplatin-induced renal tubular cell apoptosis is the activation of MAPK, including ERK (1/2), p38, and JNK 51 .Renogrit treatment decreased the activity and protein levels of the agents involved in cisplatin-induced apoptosis like caspase-3 and phosphorylated MAPKs respectively.Renogrit treatment also decreased the percentage of cells that underwent early apoptosis upon cisplatin stimulation, as observed by Annexin V-7AAD staining.All these renoprotective effects of Renogrit might be due to the presence of Quercetin as one of its major phytometabolites.It has been observed that Quercetin treatment in cisplatin-administered rats decreases the activity of caspase-3 and phosphorylation of ERK (1/2), p38, and JNK in the kidneys 52,53 .
Necroptosis, a form of programmed necrosis, is known to be stimulated in renal tubular epithelial cells.Accumulation of calcium is a major biochemical feature of necroptosis 7,54 .A cascade of kinases namely RIPK-1, RIPK-3, and MLKL are involved in regulation of cisplatin-induced necroptosis in kidney 30 .When HK-2 cells were induced with cisplatin it led to accumulation of calcium which was not the case in Renogrit-treated cells.Furthermore, the overexpression of RIPK-1, RIPK-3, and MLKL mRNA was also normalized in Renogrit-treated cells.Phenolic compounds isolated from herbs are known to reduced necroptosis mediated renal injury post cisplatin administration in mice 13 .As Renogrit is pre-dominantly composed of polyphenolic phytoconstituents it was able to keep the necroptosis activators at bay.
Treatment with high concentrations (≥ 50 µM) of cisplatin leads to a decrease in autophagy which results in the domination of apoptotic cell death in kidney cells 7 .Autophagy can enhance cytoprotection and survival of cisplatin-induced kidney cells.Cisplatin induction in HK-2 cells decreases lysosome biogenesis and autophagy of defective mitochondria (mitophagy) 9 .The process of mitophagy is tightly regulated by PINK1 which in response to loss of mitochondrial membrane potential gets accumulated at the outer membrane of the dysfunctional mitochondria where it gets dimerized, autophosphorylated, and initiates a chain of events to kickstart the process of mitophagy 32 .Cisplatin induction in HK-2 cells is also known to decrease the levels of LC3.Due to its involvement in mitophagy process a decrease in levels of LC3 indicates a defect in the mitophagy machinery 55,56 .A decrease in mitophagy leads to the accumulation of defective mitochondria due to which more ROS is generated causing DNA damage, and apoptosis indicated by cleaved-PARP1 54,57 .Renogrit treatment in cisplatin-induced HK-2 cells normalized lysosome population, enhanced mitophagy, and subsequently prevented apoptosis induced cell death.The phytochemical Quercetin present in Renogrit is known to stimulate mitophagy and prevent cell death in the in vivo models of kidney injury 58,59 .
Calcium flux, mitochondrial damage, and mitophagy dysfunction observed in cisplatin-induced kidney injury have been implicated in the NFκB and NLRP3 inflammasome pathway-mediated release of the pro-inflammatory cytokine IL-1β.An increased expression of IL-1β further triggers the activation of IL-1R inducing activation of NFκB pathway which enhances the release of other inflammatory mediators, exacerbating kidney injury 33,[60][61][62] .The LXR-α present in the kidney is one of the regulators of inflammatory response in cisplatin-induced kidney injury 34 .Renogrit decreased the IL-1β induced NFκB activation as observed from the decreased detection of SEAP released from HEK-Blue IL-1β cells.Also, in cisplatin-induced HK-2 cells, it increased the protein levels of LXR-α.These results suggest that Renogrit has anti-inflammatory properties.This might be due to the presence of several anti-inflammatory phytometabolites in Renogrit 49,63,64 .
In order to use Renogrit to protect from cisplatin-induced kidney injury in clinical settings, it becomes imperative to evaluate whether it affects the anti-cancer potential of cisplatin.Hence, the effect of Renogrit in cisplatin-induced MCF-7 (human breast cancer cell line), A549 (adenocarcinomic human alveolar basal epithelial cells), SiHa (human cervical squamous cell carcinoma cell line), and T24 (human bladder carcinoma cell line) cells were evaluated.It was observed that Renogrit treatment did not hinder the cytotoxic action of cisplatin on these tumor cells.Some previous reports suggest that plant-derived phytochemicals can exert renoprotection without affecting the chemotherapeutic activity of cisplatin on tumors 53,55,65,66 .Moreover, it was observed that in A549 cells, Renogrit enhanced the cytotoxic potential of cisplatin in a concentration-dependent manner.This effect of Renogrit can be attributed to the presence of Boerhavia diffusa L. extract as it is known to decrease cell viability of A549 cells 21 .
The effects of Renogrit were further evaluated in the in vivo model of C. elegans exposed to cisplatin.Initially, a suitable dose of cisplatin and Renogrit was screened based upon the decline in the progeny of worms.Upon selection of the suitable dose, the effects of Renogrit on cisplatin-stimulated worms were evaluated.It was found that the decrease in progeny mediated by cisplatin was reversed in response to Renogrit treatment in a dose-dependent manner.Furthermore, Renogrit treatment also perturbed the generation of ROS in worms exposed to cisplatin.Plant-based medicines are known to decrease defects in the growth and reproduction stages of nematodes, majorly in response to their antioxidant properties 67 .As expected we also found a decline in ROS generated due to Renogrit treatment in worms exposed to cisplatin.The NRF2 is activated under oxidative stress and inhibition of mitophagy in mammalian cells.Similarly, nematode homologue of NRF2, SKN-1 (SKiNhead-1) is activated during oxidative stress and inhibition of mitophagy 18,68 .Renogrit treatment reduced the activation of SKN-1 induced by cisplatin exposed SYS81 strain of C. elegans as observed from the decrease in nuclear localization of SKN-1 GFP signal in their intestine.Hence, it can be stated that Renogrit-treated worms were able to ward off oxidative stress and maintain mitochondrial turnover.
The GST-4 in C. elegans shares a sequence similarity with the GST family in humans.Overexpression of GST-4 is known to increase the stress resistance in the nematodes 69 .Renogrit treated CL2166 strain of worms showed high expression of GST-4 upon exposure of cisplatin as observed by increase in the GST-4 GFP signal.This was also observed in the in vitro findings wherein the GST levels were increased in the HK-2 cells treated with Renogrit.High levels of ROS can directly oxidize mitochondrial proteins and promote protein aggregation which leads to the accumulation of the mitochondrial chaperone, HSP-60 in the mitochondria 70 .A preclinical study by Timurkaan M et al. 71 suggests that in cisplatin-treated rats the levels of HSP-60 get upregulated in response to oxidative stress.Furthermore, they found that rats treated with the flavonoid epigallocatechin gallate decreased the protein expression of HSP-60 in the rats injected with cisplatin.Parallel to these observations when the worms of strain SJ4058 were exposed to cisplatin an increase in HSP-60 GFP signal was observed which was found to decrease in a dose-dependent manner in worms co-treated with Renogrit.These results suggest that Renogrit treatment prevents cisplatin-induced oxidative stress and subsequent mitochondrial dysfunction.
Another important mechanistic aspect that was similar to the in vitro findings on cisplatin-induced HK-2 cells was the decrease in lysosome population in the N2 strain of C. elegans.The number of lysosomes in nematodes decreased in response to cisplatin exposure as observed by LysoView 633 (pH sensitive) stained nematodes.Also, the genes involved in mitophagy namely PINK1 and PDR1 (Homologue of mammalian Parkin) were also found to decrease due to cisplatin exposure.Renogrit co-treated C. elegans were able to maintain a normal population of lysosomes and also showed an increase in genes responsible for mitophagy.Cisplatin is known to induce the overexpression of EGL-1 gene, the nematode homologue of mammalian pro-apoptotic Bcl-2 family members 72,73 .Renogrit treatment subdued the EGL-1 gene overexpression which signals that the pro-apoptotic signals generated in cells of C. elegans have been reduced.
In conclusion, the validations of the mechanistic aspects of Renogrit were performed in HK-2 cells and various strains of C. elegans.Renogrit treatment decreases the renal tubular cell injury by mitigating cisplatininduced oxidative stress, mitochondrial dysfunction, apoptosis, necroptosis, mitophagy, and inflammation by targeting multiple pathways of cell injury without affecting the anti-cancer potential of cisplatin.Similarly, in C. elegans the defects induced by cisplatin namely decrease in progeny, increase in oxidative stress, defects in mitochondria, and lowered mitophagy were mitigated by Renogrit.A summary representation of the current study is depicted in Fig. 13.Taken together, the study suggests that Renogrit has clinically relevant properties for the management of cisplatin-induced nephrotoxicity.

Phytochemical analysis of Renogrit
Renogrit (500 mg) powder (batch# CHIH/RENA/0322/2431) was dissolved in 10 ml methanol: water (80: 20 v/v), sonicated for 20 min, and centrifuged at 6000 rpm for 10 min by Sorvall ST-8R (Thermo Fisher Scientific, USA) and filtered using 0.45 µm nylon filter.An appropriate quantity of each standard was dissolved in methanol to prepare a stock solution of 1000 μg/ml from which working standards of Gallic acid (GA: 100 μg/ml), Methyl gallate (MG: 100 μg/ml), Quercetin (QT: 20 μg/ml) and Bergenin (BG: 100 μg/ml) were prepared.A HPTLC system (CAMAG, Switzerland) equipped with an automatic TLC sampler (ATS4), TLC scanner 4, TLC Visualizer, and integrated software winCATS (Version 1.4.10) was used for the HPTLC analysis on a pre-coated silica gel 60 F254 aluminium backed TLC plate.For fingerprint analysis 10 μl of each standard and 4 μl of sample were applied as 8 mm band using spray-on technique on a TLC plate.The plate was developed using CAMAG twin trough chamber pre-saturated (10 min) with mobile phase (Toluene: ethyl acetate: methanol: formic acid (5:4:1:0.2v/v/v/v).TLC plate was developed up to 70 mm, dried under warm air, and visualized under UV 254 nm.The image was documented and the same TLC was scanned at 280 nm, slit dimension was 6 × 0.45 mm, scanning speed was 20 mm/s, with data resolution of 100 μm/step.A deuterium lamp was used under absorption mode.For quantification, several concentrations of each standard solution were applied on the plate and on the basis of linearity plot concentration of specific phytochemical, concentration of the same phytochemical in Renogrit was evaluated under same chromatographic conditions.
In order to rule out any deviations in the quantity of phytometabolites characterized by HPTLC, further analysis of phytometabolites was performed by UHPLC.Three different batches of Renogrit were evaluated to generate a thorough quantitative phytochemical profile.UHPLC-based analysis of phytocompounds was evaluated on Prominence-XR UHPLC system (Shimadzu, Japan) fitted with Quaternary pump (Nexera XR LC-20AD XR), diode array detector (DAD SPD-M20 A), auto-sampler (Nexera XR SIL-20 AC XR), degassing unit (DGU-20A 5R) and column oven (CTO-10 AS VP).Separation was achieved using a Shodex C18-4E (5 µm, 4.6 × 250 mm) column subjected to binary gradient elution.The two solvents used for the analysis consisted of water comprising 0.1% acetic acid (solvent A) and acetonitrile (solvent B).Gradient programming of the solvent system was primarily at 0-5% B for 0-10 min  www.nature.com/scientificreports/ (1:1000) for 30 min.The acquisition was done in Attune NxT Flow Cytometer (Invitrogen, USA).Data were presented as mean ± S.E.M (n = 3).

Quantification of proteins by western blot
Cells were lysed with cold RIPA buffer containing (100 mM Tris, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA 0.5% sodium deoxycholate, 1% Triton X-100, and freshly supplemented with protease inhibitor (A32963, Thermo Fisher Scientific) and PhosSTOP phosphatase inhibitor (4906845001, Roche) as per manufacturer's protocol.Protein concentration in lysates was estimated by BCA method.The lysates containing 20-25 µg proteins were resolved in 10%-12% SDS-PAGE followed by transfer to a PVDF membrane (1620177, Bio-Rad).Primary antibodies used for evaluation are mentioned in Table 2.The secondary antibodies were obtained from Invitrogen, USA.Incubation time and antibody concentration were followed as per the manufacturer's protocol.Protein bands were developed using Enhanced chemiluminescent HRP substrate (WBKLS0500, Millipore, USA) in an ImageQuant LAS 500 (GE Healthcare, USA) instrument, and blots were further processed and quantified using Image Quant TL version 8.2 software (GE Healthcare, USA) and calculated according to reference bands of β-actin.

Evaluation of IL-1β activity
HEK-Blue IL-1β reporter cells co-treated with IL-1β (1 ng/ml) and Renogrit (10, 30, and 100 µg/ml) or NAC and incubated for 24 h.Post incubation the supernatant was collected and the secreted embryonic alkaline phosphatase (SEAP) levels were evaluated by QUANTI-Blue as per the manufacturer's instructions.The optical density was read at 630 nm using EnVision multimode plate reader (PerkinElmer, USA).Data were presented as mean ± S.E.M (n = 3).

Cell viability analysis of cisplatin-induced and Renogrit treated cancer cells
MCF-7, A549, SiHa, and T24 cells were seeded at a density of 10,000 cells/ well in a 96-well plate.Post-treatment the cell viability was assessed using Alamar blue dye as mentioned before.Data were presented as mean ± S.E.M (n = 3).

Nematode strains and general methods
Caenorhabditis elegans strains were obtained from the Caenorhabditis Genetics Center at the University of Minnesota, USA, and maintained as described previously 77 .The N2 (Bristol) strain was used as wild-type in all experiments and the following alleles and transgenic strains were used in this study: CL2166 (GST-4::GFP reporter strain) 78 , SJ4058 (HSP-60::GFP reporter strain) 79 , and SYS81 (SKN-1::GFP reporter strain) 80 .

Nematode brood size assessment and treatment procedure
The hatched eggs released L1 larvae which were used for exposure to various treatments.The L1 nematodes were exposed to different concentrations of Cisplatin (20-100 μg/ml) or Renogrit (3-300 μg/ml).A single L3 stage worm of each condition was isolated onto fresh petriplates with E. coli OP50.Worms were transferred to fresh plates with Cisplatin and Renogrit every other day.The progeny and number of oocytes laid were counted after 3 days.Parent worms were removed and progeny were allowed to develop for 48 h before evaluation of worms.Cisplatin (40 μg/ml) and Renogrit (10, 30, and 100 μg/ml) or NAC (4 mM) were used for further experimentation.Unless specified otherwise, L1 nematodes were pre-treated with Renogrit (10, 30, and 100 μg/ml) or NAC (4 mM) for 24 h after which the nematodes were transferred to NGM plate seeded with E. coli OP50 with or without 40 μg/ml of cisplatin exposure along with Renogrit or NAC for 4 days.Counting was done using the ZEISS Stemi 305 stereo microscope (Carl Zeiss, Germany).Data were presented as mean ± SEM (n = 5).

Figure 4 .
Figure 4. Renogrit decreased cisplatin-induced oxidative stress and mitochondrial dysfunction in HK-2 cells.(a) The reactive oxygen species (ROS) burst generated in response to HK-2 cells induced with cisplatin at human C max (15 µg/ml) was inhibited in presence of Renogrit (10-100 µg/ml).Prior to fluorescence measurements, H 2 DCFDA stained HK-2 cells were simultaneously treated with cisplatin and Renogrit or NAC (2 mM) and the converted DCF was evaluated for 120 min.(b) Renogrit treatment normalized the GST levels in HK-2 cells that were altered in response to cisplatin induction.(c) Renogrit treatment enhanced the mitochondrial membrane potential of HK-2 cells which was decreased in response to cisplatin induction as observed by ratiometric fluorescence measurements of cells stained with JC-1.Data represented as mean ± S.E.M. ### and ***p < 0.001; ## and **p < 0.01.

Figure 13 .
Figure 13.Representation of the molecular changes induced by cisplatin in HK-2 cells and in C. elegans along with the subsequent therapeutic modulation by Renogrit.
H-RIPK3Forward ATG TCG TGC GTC AAG TTA TGG Reverse CGT AGC CCC ACT TCC TAT GTTG H-RIPK1 Forward GGG AAG GTG TCT CTG TGT TTC Reverse CCT CGT TGT GCT CAA TGC AG H-MLKL Forward AGG AGG CTA ATG GGG AGA TAGA Reverse TGG CTT GCT GTT AGA AAC CTG H-NGAL Forward GAA GTG TGA CTA CTG GAT CAGGA Reverse ACC ACT CGG ACG AGG TAA CT H-β-actin Forward CAC CAA CTG GGA CGA CAT Reverse ACA GCC TGG ATA GCA ACG CE-EGL1 Forward CTA GCA GCA ATG TGC GAT GAC Reverse GGA AGC ATG GGC CGA GTA G CE-PDR1 Forward GAC TAC AAG GTG ATC TCA GCGA Reverse CGT GGC ATT TTG GGC ATC TT CE-PINK1 Forward CAA GGC GAG CCT GAA AGG A Reverse GCC GAG AAT ATT TCC CGC CA CE-ACT-1 Forward ACG ACG AGT CCG GCC CAT CC Reverse GAA AGC TGG TGG TGA CGA TGGTT

Table 1 .
Phytochemicals present in Renogrit as deciphered from chromatograms shown in Figs.1 and 2. S

Table 2 .
List of antibodies used for western blotting.All these antibodies were obtained from Invitrogen, USA except for NR1H3 (LXR-α) which was procured from Novus Biologicals, USA.

Table 3 .
Sequences of primers used for qRT-PCR of HK-2 cells (H) and C. elegans (CE).